title: Sp100 interacts with phage ΦC31 integrase to inhibit its recombination activity.
abstract: Phage ΦC31 integrase is a potential vector for the insertion of therapeutic genes into specific sites in the human genome. To understand the mechanism involved in ΦC31 integrase-mediated recombination, it is important to understand the interaction between the integrase and cellular proteins. Using a yeast two-hybrid system with pLexA-ΦC31 integrase as bait, we screened a pB42AD human fetal brain cDNA library for potential interacting cellular proteins. From the 10⁶ independent clones that were screened, 11 potential interacting clones were isolated, of which one encoded C-terminal fragment of Sp100. The interaction between Sp100 and ΦC31 integrase was further confirmed by yeast mating and co-immunoprecipitation assays. The hybridization between a ΦC31 integrase peptide array and an HEK293 cell extract revealed that residues 81RILN84 in the N-terminus of ΦC31 integrase are responsible for the interaction with Sp100. Knocking down endogenous Sp100 with Sp100-specific siRNA increased ΦC31 integrase-mediated recombination but did not impact reporter gene expression. Therefore, endogenous Sp100 may interact with ΦC31 integrase and inhibit the efficiency of ΦC31 integrase-mediated recombination.
authors.affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, P.R. China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Lin
authors.forename: Zhi-hui
authors.initials: ZH
authors.lastname: Li
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Wang
authors.forename: Gua-lan
authors.initials: GL
authors.lastname: Xu
authors.forename: Qi
authors.initials: Q
authors.lastname: Shen
authors.forename: Lin
authors.initials: L
authors.lastname: Tian
authors.forename: Jin-lun
authors.initials: JL
authors.lastname: Xue
authors.forename: Jin-zhong
authors.initials: JZ
authors.lastname: Chen
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D001435:Bacteriophages / Q000201:enzymology*; D001921:Brain / Q000196:embryology / Q000378:metabolism*; D015723:Gene Library; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D019426:Integrases / Q000378:metabolism*; D011485:Protein Binding; D034741:RNA, Small Interfering; D020798:Two-Hybrid System Techniques
chemical_list: D034961:Antigens, Nuclear; D001324:Autoantigens; D034741:RNA, Small Interfering; C066642:SP100 protein, human; D019426:Integrases